Use of manzamine compounds in anti-cancer therapeutic regimens

ABSTRACT

Manzamine compounds have been discovered to decrease cell dissociation and cell migration associated with the metastatic potential of cancer cells and a restoration of cancer cell susceptibility to agents, such as TRAIL, which can induce apoptosis. Specifically, Manzamine A has a formerly unrecognized utility in both blocking tumor cell invasion and tumor metastasis as well in restoring cancer cell susceptibility to standard chemotherapeutic agents which induce apoptosis and, therefore, has utility in treating cancer.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation application of Ser. No. 11/708,767,filed Feb. 20, 2007; which claims the benefit of U.S. provisionalapplication Ser. No. 60/775,435, filed Feb. 22, 2006, both of which areincorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

The subject matter of this application has been supported in part byU.S. Government Support under National Institutes of Health Grant No.RO1 CA 093455. Accordingly, the U.S. Government has certain rights inthis invention.

BACKGROUND OF INVENTION

Pancreatic cancer is the fourth leading cause of cancer death in theUnited States (All About Pancreatic Cancer. 2005, American CancerSociety). About 32,200 new cases will be detected and about 31,800deaths due to this disease will occur this year. The prognosis ofpancreatic cancer patients under current treatments is poor, and newdrugs to treat the disease are needed. The negative prognosis ofpancreatic cancer results from the fact that by the time a patientdisplays symptoms, the cancer has already metastasized. Treatment ofpancreatic cancer involves surgery, radiation therapy, chemotherapy or acombination of the three. The current chemotherapy for advancedpancreatic cancer is gemcitabine, a drug that inhibits DNA synthesis.Gemcitabine, while more effective than past treatments, is notsufficient to treat pancreatic cancer as shown by the lethality of thiscancer. Furthermore, there is no good therapy to treat pancreatic tumorsthat become refractory to gemcitabine (Bergenfeldt, M. and Albertsson,M. “Current state of adjuvant therapy in resected pancreaticadenocarcinoma.”. Acta Oncol, 45: 124-135, 2006). The prognosis ofpancreatic cancer patients under current treatments is poor, and newdrugs to treat the disease are needed.

Pancreatic cancer cells have high metastatic potentials and exhibitresistance to apoptosis. In pancreatic cancer, constitutivephosphorylation of Raf-MEK-ERK is a common occurrence, and it is acontributive factor to the metastatic potential of the disease bypromoting cell dissociation (Tan, X. et al. “Involvement of themitogen-activated protein kinase kinase 2 in the induction of celldissociation in pancreatic cancer”. Int J Oncol, 24: 65-73, 2004). andresistance to apoptosis (Boucher, M. J. et al. “MEK/ERK signalingpathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 andpromotes survival of human pancreatic cancer cells”. J Cell Biochem, 79:355-369, 2000). Inhibition of this pathway hinders the growth ofpancreatic cancer cells (Motomura, W. et al. “Involvement of MEK-ERKsignaling pathway in the inhibition of cell growth by troglitazone inhuman pancreatic cancer cells”. Biochem Biophys Res Commun, 332: 89-94,2005). In addition, pancreatic cancer cells also exhibit constitutiveactivation of NFκB, and its activation correlates with their metastaticpotential (Fujioka, S. et al. “Function of nuclear factor kappaB inpancreatic cancer metastasis”. Clin Cancer Res, 9: 346-354, 2003), andresistance to apoptosis. Finally, the use of a dominant-negative STAT3vector in pancreatic cancer cell lines significantly decreases theirgrowth rate (Toyonaga, T. et al. “Blockade of constitutively activatedJanus kinase/signal transducer and activator of transcription-3 pathwayinhibits growth of human pancreatic cancer”. Cancer Lett, 201: 107-116,2003), implicating this pathway in the strong metastatic potentialexhibited by pancreatic cancer cell lines. Therefore, any potentialchemotherapies that can reduce the metastatic potential or re-sensitizepancreatic cancer cells to apoptosis have the potential of being moresuccessful in the treatment of the disease than the currently availableones.

Certain cyclic alkaloid compositions, e.g., manzamines A-F derived fromextracts of the marine sponge Haliclona sp., have been found to possessuseful properties. These compounds have been described in, for example,U.S. Pat. Nos. 4,895,854; 4,895,853; and 4,895,852. Manzamines A-F havethe following structures:

BRIEF SUMMARY

The present invention pertains to the use of manzamine compounds for thetreatment of cancer. In a preferred embodiment, a manzamine compound isused as an adjuvant or in combination therapy with existingchemotherapeutics in an anti-cancer therapeutic regimen. Specificallyexemplified herein is the use of manzamine A, which has been found to beparticularly effective because of its anti-cancer activity and lowtoxicity.

As described herein, the invention also comprises pharmaceuticalcompositions, e.g. anti-cancer compositions, containing as an activeingredient an effective amount of one or more manzamine compounds asdescribed herein and a non-toxic, pharmaceutically acceptable carrier ordiluent. The pharmaceutical compositions of the subject invention canfurther comprise other active compounds. As described herein, theinvention further comprises novel methods of use of the manzamines, e.g.anti-cancer methods.

In accordance with the subject invention, methods for inhibiting cancercomprise administering to a human or animal in need of such treatment aneffective amount of the pharmaceutical compositions described herein. Ina preferred embodiment the manzamine compound is used as part of atreatment for pancreatic cancer. In a further preferred embodiment, themanzamine is used in conjunction with a compound that induces apoptosis.

Specifically exemplified herein is the ability of manzamine A todecrease cell disassociation associated with pancreatic cancer cells,its ability to abrogate cell migration which is associated with themetastatic potential of pancreatic cancer cells, and restoration ofcancer cell susceptibility to agents, such as TNF-relatedapoptosis-inducing ligand (TRAIL), that can induce apoptosis. Theseeffects resemble those caused by inhibitors of pMEK.

In accordance with the subject invention, manzamine A has a formerlyunrecognized utility in both blocking tumor cell invasion and tumormetastasis as well in restoring cancer cell susceptibility to agentsthat induce apoptosis and therefore, in one embodiment, has utility asan adjuvant to, or in combination with, existing cancer therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the average of four experiments to determine thecytotoxicity of manzamine A against pancreatic cancer cells.

FIG. 2 shows that manzamine A lowers the resistance to apoptosisexhibited by pancreatic cancer cells.

FIG. 3 shows abrogation of cell dissociation by Manzamine A.

FIG. 4 shows abrogation of cell migration by Manzamine A.

DETAILED DISCLOSURE

In accordance with the subject invention, methods for inhibiting cancerare provided wherein cancer cells are contacted with an effective amountof a manzamine compound. In a preferred embodiment, a manzamine compoundis used as an adjuvant or in combination therapy with existingchemotherapeutics in an anti-cancer therapeutic regimen. Specificallyexemplified herein is the use of manzamine A, which has been found to beparticularly effective because of its anti-cancer activity and lowtoxicity.

Thus, the subject invention pertains to novel uses as anti-cancer agentsof manzamine compounds and compositions comprising the manzaminecompounds. The manzamine compounds of the subject invention can be usedto inhibit metastasis of cancer cells and restore sensitivity toprogrammed cell death.

In one embodiment, the subject invention pertains to the use ofcompounds having the following General Structure (I):

wherein X¹, X², X³, X⁴, X⁵, and X⁶ are, independently, a hydrogen,halogen, hydroxy, lower alkoxy, lower acyloxy, or lower mono or dialkylamino group; R¹ is hydrogen, lower alkyl, or lower acyl group; R² ishydrogen, hydroxy, lower alkoxy, or lower acyloxy group.

In a preferred embodiment of the invention, the invention pertains tothe cancer chemotherapeutic use as either an adjuvant or in combinationtherapy of manzamine A having the following structure:

Further embodiments of the subject invention pertain to the cancerchemotherapeutic use as either an adjuvant or in combination therapy ofcompounds having General Structures (II)-(IV):

wherein X¹, X², X³, X⁴, X⁵, and X⁶ are the same or different and are ahydrogen, halogen, hydroxyl, lower alkoxy, lower acyloxy, thiol, loweralkylthiol, nitro, amino, lower alkylsulfonyl, aminosulfonyl, hydroxysulfonyl (—SO₃H), lower acylamino, lower alkyl, or lower monoalkyl- ordialkyl-amino group; R¹ and R² are the same or different and are ahydrogen, lower alkyl, or lower acyl group; and Y is a hydrogen,hydroxyl, lower alkoxy, or lower acyloxy group.

In more specific embodiments of the invention, the invention comprisesthe the cancer chemotherapeutic use as either an adjuvant or incombination therapy of the compounds designated as manzamine B, C, or Dof the formulae:

Further embodiments of the subject invention utilize General StructureV:

wherein R is a hydrogen, halogen, hydroxy, or lower acyloxy group; and Xis a double bonded oxygen, or is the same or different and is any two ofa hydrogen, hydroxy, lower alkyl, lower alkoxy, or lower acyloxy groupwherein said lower alkyl, alkoxy, or acyloxy groups have preferably,from 1 to 5 carbon atoms.

In one embodiment, the subject invention concerns manzamines E and Fwhich have the following structures:

In other embodiments of the invention, the double bonds in thecomposition of General Structures (I)-(V) are partially or fullyreduced. In further embodiments of the invention, the composition is amineral acid (e.g., HCl, H₂SO₄, H₃PO₄, HNO₃, etc.) or organic salt ofcompositions according to the General Structures.

Methods for obtaining these compounds are described in, for example,U.S. Pat. Nos. 4,895,852; 4,895,853; and 4,895,854, which are hereinincorporated in their entirety by reference thereto.

Skilled chemists having the benefit of the instant disclosure canreadily use procedures to prepare the subject compounds. In carrying outsuch operations, suitable filtration, chromatographic and otherpurification techniques can be used. These techniques could include, forexample, reversed phase (RPLC), column, vacuum flash, medium pressure(MPLC) and high performance liquid chromatography (HPLC) with a suitablecolumn such as silica gel, Sephadex LH-20, ammonia-treated silica gel,bonded phase RP-18, RP-8 and amino columns. Such columns are eluted withsuitable solvents such as heptane, ethyl acetate, methylene chloride,methanol, isopropanol, acetonitrile water, trifluoroacetic acid (TFA)and various combinations thereof.

Most preferably, the invention comprises a method for the anti-cancertreatment of a human in need of such treatment, i.e., a human hostingcancer cells, including breast, renal, colon, liver, pancreatic,uterine, or lung tumor cells, or leukemia cells.

In a specific embodiment of the subject invention, manzamine A has beendiscovered to abrogate cell dissociation as well as the ability ofpancreatic cells to migrate through extracellular matrices, bothcharacteristics that correlate with the high metastatic potential ofpancreatic cancer cells Furthermore, manzamine A has been shown toabrogate the resistance to TRAIL-mediated apoptosis, which causes thefailure of many current therapies. Effects were achieved with manzamineA at a fifth of the dose needed for U0126, a well-characterized MEKinhibitor, to produce similar results. Moreover, unlike U0126 which isfairly unstable in solution, manzamine A is stable and shows littlecytotoxicity at small doses. In view of these results, in one embodimentof the subject invention, manzamine A is advantageous for use incombination therapy in the treatment of pancreatic cancer.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Materials and Methods

Reagents. Manzamine A was obtained from the Harbor Branch OceanographicInstitute (HBOI) pure compound library. The material was originallyisolated from a sponge of the genus Haliclona as described in U.S. Pat.No. 4,895,854. The manzamine A stock solution was at 5 mM concentrationin methanol. U0126, a known pMEK inhibitor used as a positive control inthe assays, was purchased from Calbiochem, San Diego, Calif.

Cell Culture Conditions for the AsPC-1 pancreatic adenocarcinoma cellline. AsPC-1, a pancreatic adenocarcinoma cell line, was obtained fromATCC, grown, aliquotted and frozen stocks were maintained in liquidnitrogen. Aliquots of the stock cells were thawed and grown in RPMI-1640supplemented with 10% Fetal Bovine Serum, 0.11 mg/ml Sodium Pyruvate,4.5 g/L D-glucose, 18 mM HEPES Buffer, 100 U/ml penicillin G sodium, 100μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B, 2 mM L-glutamineand 50 μg/ml gentamicin (Complete RPMI). Cells were maintained in ahumidified incubator at 37° C. and 5% CO₂.

Cell viability assays. A3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT)based assay was used to determine cell viability. For the MTT assay,12,000 AsPC-1 cells were plated into a 96-well tissue culture plate at avolume of 200 μl/well. Cells were allowed to adhere for 24 hours. At theend of this incubation, 100 μl of medium were removed from each testwell and 100 μl of medium containing treatment were added. Treatmentconsisted of 5 μM or 50 μM of Manzamine A, media alone, media withmethanol, or 20 μg/ml U0126. The cells were then incubated for 48 hoursat 37° C. and 5% CO₂. After this incubation, 75 μl 5 mg/ml MTT wereadded to each well. The cells were then incubated for 3 hours at 37° C.The plates were centrifuged for 10 minutes at 800 rpm. The supernatantwas removed and 200 μl acidified isopropyl alcohol (1:500 solution ofhydrochloric acid to isopropanol) was added to each well. The plateswere shaken for 15 minutes. The absorbencies of these solutions weremeasured at 570 nm with a plate reader (NOVOstar, BMG Labtech Inc.,Durham, N.C.). The resulting absorbencies were plotted using MicrosoftExcel.

TRAIL induced apoptosis. 12,000 AsPC-1 cells were plated on a 96-welltissue culture plate at a volume of 200 μl/well, allowing for twelvereplicate wells per treatment. Cells were allowed to adhere for 24hours. At the end of this incubation, 100 μl of medium were removed fromeach test well and 100 μl of medium containing treatment were added.Treatment consisted of media alone, media with methanol, 10 μM ManzamineA or 20 μg/m1 U0126. The cells were then incubated for 48 hours at 37°C. and 5% CO₂. After this incubation, half of the wells of eachtreatment were incubated with 100 ng/ml Super Killer TRAIL (Alexis, SanDiego, Calif.) for 18 hours at 37° C. and 5% CO₂. At the end of thisincubation, 75 μl of a 5 mg/ml MTT solution were added to each well. Thecells were then incubated for 3 hours at 37° C. The plates werecentrifuged for 10 minutes at 800 rpm. The supernatant was removed and200 μl acidified isopropyl alcohol (1:500 solution of hydrochloric acidto isopropanol) were added to each well. The plates were shaken for 15minutes. The absorbencies of these solutions were measured at 570 nmwith a plate reader (NOVOstar, BMG Labtech Inc., Durham, N.C.). Theresulting absorbencies were plotted using Microsoft Excel.

In Vitro Cell Invasion Assay. The invasiveness of AsPC-1 cells alone,treated with our test compounds or treated with U0126 as a positivecontrol will be evaluated in 24-well transwell chambers, as described byFu et al. “Effects of raf kinase inhibitor protein expression onsuppression of prostate cancer metastasis”. J Natl Cancer Inst, 95:878-889, 2003. Briefly, the upper and lower culture compartments of eachwell are separated by polycarbonate membranes (8-μm pore size). Todetermine baseline migration, 2.5×10⁴ cells in 0.5 mL of complete mediumcontaining 5% FBS are placed into the upper compartment of uncoatedwells (BD Discovery Labware, San Diego, Calif.), and 0.75 mL of completemedium containing 10% FBS are placed into the lower compartment. Inparallel, to assess the ability of the same cells to penetrate acollagen matrix, the experiment is repeated using upper compartmentscoated with 100 μg/cm² of collagen matrix (BD Discovery Labware, SanDiego, Calif.). The transwell chambers are incubated for 24 hours at 37° C. in 95% air and 5% CO₂. Cell penetration through the membrane isdetected by staining the cells on the porous membrane with a Diff-Quikstain kit' observing under the microscopic and capturing images with adigital camera.

Statistical Analysis of Results. Statistical analysis of the data setsto determine mean, standard deviation, and standard error of the meanwas performed using Microsoft Excel. Data sets were compared using theStudent's T Test. A p value≦0.05 was considered significant. Outlierswere detected through the Grubbs Test.

The scope of the invention is not limited by the specific examples andsuggested procedures and uses related herein since modifications can bemade within such scope from the information provided by thisspecification to those skilled in the art.

The examples which follow are not meant to be fully inclusive of allforms of cancer which can be treated with manzamine A nor is itinclusive of all chemotherapeutic agents with which manzamine A could beused in combination for the treatment of cancer.

EXAMPLE 1 Measurement of Morphological Changes Induced in AsPC-1 Cellsby Manzamine A

The high metastatic potential exhibited by pancreatic cancer cells is inpart due to their ability to dissociate from other pancreatic cells andgrow in single cell formation. An observation was made that inhibitingthe constitutive activation of MEK abrogates cell dissociation (Tan, X.et al., 2004 “Involvement of the mitogen-activated protein kinase kinase2 in the induction of cell dissociation in pancreatic cancer” Int JOncol 24(1):65-73), a precursor step in metastasis. Therefore, todetermine if manzamine A caused the abrogation of cell dissociation,AsPC-1 cells were treated for 48 hours with media alone, mediacontaining methanol, 52.6 μM U0126 or 1.25, 2.5, 5, 10 μM Manzamine A.At the end of this incubation, cells were visualized with the microscopeand photographed with a digital camera. Manzamine A caused abrogation ofcell dissociation similar to that caused by pMEK inhibitors.

The abrogation was stronger at the 5 and 10 μM dose of Manzamine A.Thus, in one embodiment, the cancers that can be treated in accordancewith the subject invention are those for which inhibition ofphosphorylated mitogen-activated protein kinase kinase 2 (p-MEK) causesthe cancer to be less likely to proliferate and/or metastasize, and/orwhich either directly or indirectly makes the cancer cells more prone toapoptosis (all of which are collectively referred to herein as“inhibiting the growth” of cancer cells).

EXAMPLE 2 Measurement of the Ability of AsPC-1 Cells to Migrate Througha Matrix

Another contributor to the high metastatic potential of pancreaticcancer cells is their ability to migrate through extracellular matrices,which normal cells are unable to do. To determine if manzamine Aprevented cell migration, AsPC-1 cells were treated for 48 hours withmedia alone, media containing methanol, 52.6 μM U0126 or 1.25, 2.5, 5,10 μM Manzamine A and subjected to an in vitro cell invasion assay asdescribed in the materials and methods. At the end of this incubation,cells were stained, visualized with the microscope and photographed witha digital camera. Manzamine A at doses of 5 and 10 μM prevented cellmigration similar to that caused by the pMEK inhibitor U0126.

EXAMPLE 3 Measurement of Sensitivity of AsPC-1 Cells to TRAIL-inducedApoptosis when Treated with Manzamine A

To determine if treatment with manzamine A sensitized pancreatic cancercells to TRAIL-mediated apoptosis, cells were treated with media alone,media containing methanol, 52.6 μM U0126, and 10 μM Manzamine A for 48hours as per the protocol listed in the materials and methods. At theend of this treatment, half of the cells were further treated with 100mg/ml super killer TRAIL. Treatment with 10 μM manzamine A or theinhibitor U0126 induced some cytotoxicity. Treatment with super killerTRAIL induced some apoptosis in all the cells regardless of treatment.However, when the cells were treated with super killer TRAIL antibody inaddition to the inhibitors, pre-treatment with manzamine A caused thegreatest level of TRAIL-mediated cytotoxicity.

EXAMPLE 4 Formulation and Administration

The compounds of the invention are useful for various non-therapeuticand therapeutic purposes. It is apparent from the testing that thecompounds of the invention are effective for decreasing metastaticpotential and resistance to apoptosis similar to known pMEK inhibitors.Because of the properties of the compounds, they are useful to preventunwanted cell growth in a wide variety of settings including in vitrouses. As disclosed herein, they are also useful for treating cancercells in animals and humans.

Therapeutic application of the compounds and compositions containingthem can be accomplished by any suitable therapeutic method andtechnique presently or prospectively known to those skilled in the art.

The dosage administration to a host in the above indications will bedependent upon the identity of the cancer cells, the type of hostinvolved, its age, weight, health, kind of concurrent treatment, if any,frequency of treatment, and therapeutic ratio.

The compounds of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin describesformulations which can be used in connection with the subject invention.In general, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive compound(s) is combinedwith a suitable carrier in order to facilitate effective administrationof the composition.

In accordance with the invention, pharmaceutical compositionscomprising, as an active ingredient, an effective amount of one or moreof the new compounds and one or more non-toxic, pharmaceuticallyacceptable carrier or diluent. Examples of such carriers for use in theinvention include ethanol, dimethyl sulfoxide, glycerol, silica,alumina, starch, and equivalent carriers and diluents.

To provide for the administration of such dosages for the desiredtherapeutic treatment, new pharmaceutical compositions of the inventionwill advantageously comprise between about 0.1% and 45%, and especially,1 and 15%, by weight of the total of one or more of the new compoundsbased on the weight of the total composition including carrier ordiluent. Illustratively, dosage levels of the administered activeingredients can be: intravenous, 0.01 to about 20 mg/kg;intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 to about100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 to about200 mg/kg, and preferably about 1 to 100 mg/kg; intranasal instillation,0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20 mg/kg of animal(body) weight.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

We claim:
 1. A method for inducing apoptosis in pancreatic cancer cellswherein said method comprises administering, to the cancer cells, aneffective amount of manzamine Λ and TNF-related apoptosis-inducingligand.